Method to prevent accelerated atherosclerosis using (sRAGE) soluble receptor for advanced glycation endproducts

ABSTRACT

The present invention provides for a method to prevent accelerated atherosclerosis in a subject predisposed thereto which comprises administering to the subject a polypeptide derived from soluble receptor for advanced glycation endproduct in an amount effective to prevent accelerated atherosclerosis in the subject. The present invention also provides for a method to prevent a macrovessel disease in a subject predisposed thereto which comprises administering to the subject a polypeptide derived from soluble receptor for advanced glycation endproduct in an amount effective to prevent macrovessel disease in the subject.

This application is a continuation of U.S. Ser. No. 08/905,709, filedAug. 5, 1997, the contents of which are hereby incorporated by referencein their entirety into the present application.

The invention disclosed herein was made with Government support underNIH Grants No. HL56881 and AG00602 from the Department of Health andHuman Services. Accordingly, the U.S. Government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byauthor and date within the text. Full citations for these publicationsmay be found listed alphabetically at the end of the specificationimmediately following the Sequence Listing and before the claims. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art as known to those skilled therein as ofthe date of the invention described and claimed herein.

Ischemic heart disease is a leading cause of morbidity and mortality inthe general population, but especially in patients with diabetes. Theprevalence of coronary artery disease is as high as 55% in adultpatients with diabetes (Robertson and Strong, 1968). Indeed, data fromthe Framingham Heart Study demonstrate that mortality fromcardiovascular disease in non-insulin dependent diabetes (NIDDM) is morethat doubled in diabetic men and more than quadrupled in diabetic womenwhen compared to nondiabetic control subjects (Kannel and McGee, 1979).In addition to increased prevalence, studies have shown thatatherosclerosis in diabetic patients is clearly more accelerated andextensive. In one autopsy series, for example, patients with diabeteswere found to have more severe disease of the left anterior descendingcoronary artery (Waller et al., 1980), a higher incidence of two andthree-vessel disease (Crall and Roberts, 1978), and a greaterdiffuseness of distribution of atherosclerotic lesions (Hamby et al.,1976). The findings were confirmed by coronary angioplasty insymptomatic patients (Pyorala et al., 1978).

The reasons for accelerated atherosclerosis in the setting of diabetesare numerous. However, even after correction for dyslipidemia,hypertension and obesity, multivariate analysis studies have indicatedthat diabetic patients have an excess risk of cardiovascular diseaserelative to nondiabetic subjects (Kannel and McGee, 1979). For example,in the Nurses' Health Study of 1,500 diabetic subjects among a total of115,000 women, the incidence of cardiovascular disease was 5-fold higherin the diabetic subjects regardless of their levels of cholesterol(Manson et al., 1991). The data suggest that factors unique to thediabetic population play an important role.

SUMMARY OF THE INVENTION

The present invention provides for a method to prevent acceleratedatherosclerosis in a subject predisposed thereto which comprisesadministering to the subject a polypeptide derived from soluble receptorfor advanced glycation endproduct in an amount effective to preventaccelerated atherosclerosis in the subject. The present invention alsoprovides for a method to prevent a macrovessel disease in a subjectpredisposed thereto which comprises administering to the subject apolypeptide derived from soluble receptor for advanced glycationendproduct in an amount effective to prevent macrovessel disease in thesubject.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B. Gross appearance of the proximal aorta of apolipoproteinE(0) mice under dissection microscopy. Aortic specimens were subjectedto retrograde injection of methylene blue in apolipoprotein E (0) micewith diabetes (16 wk old mice; 10 wks diabetes, FIG. 1A) or age-matchednondiabetic controls (16 wk old, FIG. 1B).

FIG. 2. Treatment of diabetic apolipoprotein E (0) mice with sRAGEsuppresses accelerated atherosclerosis. Apolipoprotein E (0) mice wererendered diabetic with stz. After 2 weeks of diabetes, mice were treatedwith either sRAGE (20 μg/day, intraperitoneally) or equimolar amounts ofmouse serum albumin (40 μg/day, intraperitoneally) for 6 more wks. Meanlesion area in diabetic mice treated with sRAGE, 150,046±18,549 μm² wassignificantly less than that observed in mice treated with mouse serumalbumin, 271,008±16,721 μm², p, 0.02.

FIGS. 3A, 3B. Gross appearance of the proximal aorta of diabeticapolipoprotein E (0) mice treated with mouse serum albumin(left panel)or soluble mouse RAGE(right panel) under dissection microscopy.Apolipoprotein E (0) mice were rendered diabetic with stz. After 2 wksof diabetes, mice were treated with either sRAGE (20 μg/day,intraperitoneally) or equimolar amounts of mouse serum albumin(40μg/day, intraperitoneally) for 6 more wks. Gross inspection of theproximal aorta revealed nearly complete absence of lesions in the secondand third branches of the proximal aorta in mice treated with sRAGEcompared to those treated with mouse serum albumin. Marked decrease inlesions at the first branch point and at the arch of the aorta were alsoobserved in sRAGE-treated mice.

FIGS. 4A-B. Nucleotide and Amino Acid Sequence of Bovine and Human RAGE.The bovine (FIG. 4A) and human (FIG. 4B) genes were sequenced by thedideoxy chain termination method. Potential N-linked glycosylation sitesare indicated by boxed sequences, the putative polyadenylation sites areshown with bold underlining, and sequences matching the sequenced bovinepeptides are indicated by light underlining. The following amino acidresidues from the underlined peptide sequences were not determined byprotein sequencing: all Cys (C) and Trp (W), Asn25 (N25) and Glu50(E50). The bovine nucleotide amino acid sequence is SEQ. ID. NO:1. Thebovine amino acid sequence is SEQ. ID. NO:2. The human nucleotidesequence is SEQ. ID. NO:3. The human amino acid sequence is SEQ. ID.NO:4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a method to prevent acceleratedatherosclerosis in a subject predisposed thereto which comprisesadministering to the subject a polypeptide derived from soluble receptorfor advanced glycation endproduct in an amount effective to preventaccelerated atherosclerosis in the subject.

The subject may be a mammal. The mammal may be a human. The subject maybe a diabetic subject. The subject may be suffering from anapolipoprotein deficiency, or from hyperlipidemia. The hyperlipidemiamay be hypercholesterolemia or hypertriglyceridemia. The subject mayhave a glucose metabolism disorder. The subject may be an obese subject.

In one embodiment of the invention, the polypeptide may comprise atleast a portion of naturally occuring soluble receptor for advancedglycation endproduct. The polypeptide may comprise a “V” domain ofnaturally occuring soluble receptor for advanced glycation endproduct.The polypeptide may comprise a 10 kilodalton domain of naturallyoccuring soluble receptor for advanced glycation endproduct.

The polypeptide may comprise a sequence less than or equal to 20 aminoacids in length which sequence is within the sequence of the naturallyoccuring soluble receptor for advanced glycation endproduct. Forexample, the sequence may be 5 amino acids in length, 3 amino acids inlength, 8 amino acids in length or 11 amino acids in length. The lengthmay also be any other length between 2 and 20 amino acids. In oneembodiment of the invention, the length may be one amino acid.

The polypeptide may be a peptidomimetic, a synthetic polypeptide or apolypeptide analog. The polypeptide may be a non-natural polypeptidewhich has chirality not found in nature, i.e. D-amino acids or L-aminoacids.

In another embodiment of the present invention, the method may furthercomprise administering to the subject a pharmaceutically acceptablecarrier during the administration of the polypeptide. The administrationmay comprise intralesional, intraperitoneal, intramuscular orintravenous injection; infusion; liposome-mediated delivery; or topical,nasal, oral, ocular or otic delivery.

The polypeptide may be delivered hourly, daily, weekly, monthly, yearly(e.g. in a time release form) or as a one time delivery. The deliverymay be continuous delivery for a period of time, e.g. intravenousdelivery.

The effective amount of the polypeptide may comprise from about 0.000001mg/kg body weight to about 100 mg/kg body weight. In one embodiment, theeffective amount may comprise from about 0.001 mg/kg body weight toabout 50 mg/kg body weight. In another embodiment, the effective amountmay range from about 0.01 mg/kg body weight to about 10 mg/kg bodyweight. The actual effective amount will be based upon the size of thepolypeptide, the biodegradability of the polypeptide, the bioactivity ofthe polypeptide and the bioavailability of the polypeptide. If thepolypeptide does not degrade quickly, is bioavailable and highly active,a smaller amount will be required to be effective. The effective amountwill be known to one of skill in the art; it will also be dependent uponthe form of the polypeptide, the size of the polypeptide and thebioactivity of the polypeptide. One of skill in the art could routinelyperform empirical activity tests for a polypeptide to determine thebioactivity in bioassays and thus determine the effective amount.

The present invention also provides for a method to prevent amacrovessel disease in a subject predisposed thereto which comprisesadministering to the subject a polypeptide derived from soluble receptorfor advanced glycation endproduct in an amount effective to preventmacrovessel disease in the subject.

The subject may be a human or an animal. The subject may be a diabeticsubject. The subject may be suffering from an apolipoprotein deficiency.The subject may be suffering from hyperlipidemia. The hyperlipidemia maybe hypercholesterolemia or hypertriglyceridemia. The subject may have aglucose metabolism disorder. The subject may be an obese subject.

In one embodiment of the invention, the polypeptide comprises at least aportion of naturally occuring soluble receptor for advanced glycationendproduct (RAGE). The polypeptide may comprise a “V” domain ofnaturally occuring soluble receptor for advanced glycation endproduct.

The polypeptide may comprise a 10 kilodalton domain of naturallyoccuring soluble receptor for advanced glycation endproduct. Thepolypeptide may comprises less than or equal to 20 amino in length whichsequence is within the sequence of the naturally occuring solublereceptor for advanced glycation endproduct.

The polypeptide may be a peptidomimetic, a synthetic polypeptide or apolypeptide analog.

In another embodiment of the present invention, the method may furthercomprise administering a pharmaceutically acceptable carrier to thesubject during the administration of the polypeptide.

The administration may comprise intralesional, intraperitoneal,intramuscular or intravenous injection; infusion; liposome-mediateddelivery; or topical, nasal, oral, ocular or otic delivery.

The sRAGE polypeptide may be administered hourly, daily, weekly,monthly, yearly (e.g. in a time release form) or as a one time delivery.The delivery or administration may be continuous delivery for a periodof time, e.g. intravenous delivery.

The following abbreviations are used herein: AGE—advanced glycationendproduct(s); RAGE—receptor for advanced glycation endprocut(s);sRAGE—soluble receptor for advanced glycation endproduct(s).

The polypeptide may be a peptide, a peptidomimetic, a syntheticpolypeptide, a derivative of a natural polypeptide, a modifiedpolypeptide, a labelled polypeptide, or a polypeptide which includesnon-natural peptides. The peptidomimetic may be identified fromscreening large libraries of different compounds which arepeptidomimetics to determine a compound which is capable of preventingaccelerated atherosclerosis in a subject predisposed thereto.

The polypeptide may be a derivative of soluble receptor for advancedglycation end product (sRAGE). The polypeptide may be a solubleextracellular portion of a receptor for advanced glycation end product,an antibody or portion thereof, wherein the antibody is capable ofspecifically binding to the receptor for advanced glycation endproduct.The antibody may be a monoclonal antibody or a polyclonal antibody. Aportion of the antibody may be a Fab or a complementarity determiningregion or a variable region. The polypeptide may be capable ofspecifically binding to the amyloid-β peptide. The polypeptide may bindto the amyloid-β peptide at the site where the receptor for advancedglycation end product interacts.

In addition to naturally-occurring forms of polypeptides derived fromsRAGE, the present invention also embraces other sRAGE polypeptides suchas polypeptide analogs of sRAGE. Such analogs include fragments ofsRAGE. Following the procedures of the published application by Alton etal. (WO 83/04053), one can readily design and manufacture genes codingfor microbial expression of polypeptides having primary conformationswhich differ from that herein specified for in terms of the identity orlocation of one or more residues (e.g., substitutions, terminal andintermediate additions and deletions). Alternately, modifications ofcDNA and genomic genes can be readily accomplished by well-knownsite-directed mutagenesis techniques and employed to generate analogsand derivatives of sRAGE polypeptide. Such products share at least oneof the biological properties of sRAGE but may differ in others. Asexamples, products of the invention include those which areforeshortened by e.g., deletions; or those which are more stable tohydrolysis (and, therefore, may have more pronounced or longerlastingeffects than naturally-occurring); or which have been altered to deleteor to add one or more potential sites for O-glycosylation and/orN-glycosylation or which have one or more cysteine residues deleted orreplaced by e.g., alanine or serine residues and are potentially moreeasily isolated in active form from microbial systems; or which have oneor more tyrosine residues replaced by phenylalanine and bind more orless readily to target proteins or to receptors on target cells. Alsocomprehended are polypeptide fragments duplicating only a part of thecontinuous amino acid sequence or secondary conformations within sRAGE,which fragments may possess one property of sRAGE and not others. It isnoteworthy that activity is not necessary for any one or more of thepolypeptides of the invention to have therapeutic utility or utility inother contexts, such as in assays of sRAGE antagonism. Competitiveantagonists may be quite useful in, for example, cases of overproductionof sRAGE.

Of applicability to polypeptide analogs of the invention are reports ofthe immunological property of synthetic peptides which substantiallyduplicate the amino acid sequence extant in naturally-occurringproteins, glycoproteins and nucleoproteins. More specifically,relatively low molecular weight polypeptides have been shown toparticipate in immune reactions which are similar in duration and extentto the immune reactions of physiologically-significant proteins such asviral antigens, polypeptide hormones, and the like. Included among theimmune reactions of such polypeptides is the provocation of theformation of specific antibodies in immunologically-active animals[Lerner et al., Cell, 23, 309-310 (1981); Ross et al., Nature, 294,654-658 (1981); Walter et al., Proc. Natl. Acad. Sci. USA, 78, 4882-4886(1981); Wong et al., Proc. Natl. Sci. USA, 79, 5322-5326 (1982); Baronet al., Cell, 28, 395-404 (1982); Dressman et al., Nature, 295, 185-160(1982); and Lerner, Scientific American, 248, 66-74 (1983). See also,Kaiser et al. [Science, 223, 249-255 (1984)] relating to biological andimmunological properties of synthetic peptides which approximately sharesecondary structures of peptide hormones but may not share their primarystructural conformation.

The polypeptide of the present invention may be a peptidomimeticcompound which may be at least partially unnatural. The peptidomimeticcompound may be a small molecule mimic of a portion of the amino acidsequence of sRAGE. The compound may have increased stability, efficacy,potency and bioavailability by virtue of the mimic. Further, thecompound may have decreased toxicity. The peptidomimetic compound mayhave enhanced mucosal intestinal permeability. The compound may besynthetically prepared. The compound of the present invention mayinclude L-,D- or unnatural amino acids, alpha, alpha-disubstituted aminoacids, N-alkyl amino acids, lactic acid (an isoelectronic analog ofalanine). The peptide backbone of the compound may have at least onebond replaced with PSI-[CH═CH] (Kempf et al. 1991). The compound mayfurther include trifluorotyrosine, p-Cl-phenylalanine,p-Br-phenylalanine, poly-L-propargylglycine, poly-D,L-allyl glycine, orpoly-L-allyl glycine.

One embodiment of the present invention is a peptidomimetic compoundhaving the biological activity of preventing accelerated athersclerosisin a subject wherein the compound has a bond, a peptide backbone or anamino acid component replaced with a suitable mimic. Examples ofunnatural amino acids which may be suitable amino acid mimics includeβ-alanine, L-α-amino butyric acid, L-γ-amino butyric acid, L-α-aminoisobutyric acid, L-ε-amino caproic acid, 7-amino heptanoic acid,L-aspartic acid, L-glutamic acid, cysteine (acetamindomethyl),N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methioninesulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine,N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine,Boc-hydroxyproline, Boc-L-thioproline. (Blondelle, et al. 1994; Pinilla,et al. 1995).

The subject may be a mammal or non-mammal. The subject may be a human.The subject may be a mouse, a cow, a monkey, a horse, a pig, or a dog.The subject may be a diabetic subject. The subject may be suffering froman apolipoprotein deficiency. The subject may have a glucose metabolismdisorder. The subject may be an obese subject. The subject may havegenetically-mediated or diet-induced hyperlipidemia. AGEs form inlipid-enriched environments even in euglycemia.

The administration in this embodiment may be intralesional,intraperitoneal, intramuscular or intravenous injection; infusion;liposome-mediated delivery; topical, nasal, oral, anal, ocular or oticdelivery. The administration may be constant for a certain period oftime or periodic and at specific intervals.

The carrier may be a diluent, an aerosol, a topical carrier, an aqeuoussolution, a nonaqueous solution or a solid carrier.

In the practice of any of the methods of the invention or preparation ofany of the pharmaceutical compositions a therapeutically effectiveamounts is an amount which is capable of preventing acceleratedatherosclerosis in a subject predisposed thereto. Accordingly, theeffective amount will vary with the subject being treated, as well asthe condition to be treated. For the purposes of this invention, themethods of administration are to include, but are not limited to,administration cutaneously, subcutaneously, intravenously, parenterally,orally, topically, or by aerosol.

As used herein, the term “suitable pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, water, emulsions such as anoil/water emulsion or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. An example of anacceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid®.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients.

This invention also provides for pharmaceutical compositions includingtherapeutically effective amounts of polypeptide compositions andcompounds, capable of preventing accelerated atherosclerosis in asubject by inhibiting the binding of an amyloid-β peptide with areceptor for advanced glycation endproduct, together with suitablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions may be liquids or lyophilized or otherwisedried formulations and include diluents of various buffer content (e.g.,Tris-HCl., acetate, phosphate), pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, detergents (e.g.,Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents(e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzylalcohol, parabens), bulking substances or tonicity modifiers (e.g.,lactose, mannitol), covalent attachment of polymers such as polyethyleneglycol to the compound, complexation with metal ions, or incorporationof the compound into or onto particulate preparations of polymericcompounds such as polylactic acid, polglycolic acid, hydrogels, etc, oronto liposomes, micro emulsions, micelles, unilamellar or multi lamellarvesicles, erythrocyte ghosts, or spheroplasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance of the compound or composition.The choice of compositions will depend on the physical and chemicalproperties of the compound capable of preventing acceleratedatherosclerosis in a subject predisposed thereto.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.,poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

Portions of the polypeptide or composition of the invention may be“labeled” by association with a detectable marker substance (e.g.,radiolabeled with ¹²⁵I or biotinylated) to provide reagents useful indetection and quantification of compound or its receptor bearing cellsor its derivatives in solid tissue and fluid samples such as blood,cerebral spinal fluid or urine.

When administered, compounds are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive compounds may by required to sustaintherapeutic efficacy. Compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified compounds(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987).Such modifications may also increase the compound's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound adducts less frequently or in lower doses thanwith the unmodified compound.

Attachment of polyethylene glycol (PEG) to compounds is particularlyuseful because PEG has very low toxicity in mammals (Carpenter et al.,1971). For example, a PEG adduct of adenosine deaminase was approved inthe United States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicityand antigenicity of heterologous compounds. For example, a PEG adduct ofa human protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The polypeptide or composition of the present invention may bedelivered in a microencapsulation device so as to reduce or prevent anhost immune response against the polypeptide or against cells which mayproduce the polypeptide. The polypeptide or composition of the presentinvention may also be delivered microencapsulated in a membrane, such asa liposome.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

Clinical Aspects

In one embodiment of the present invention, the subject may be sufferingfrom clinical aspects as described hereinbelow and as further describedin Harper's Biochemistry, R. K. Murray, et al. (Editors) 21st Edition,(1988) Appelton & Lange, East Norwalk, Conn. Such clinical aspects maypredispose the subject to atherosclerosis or to acceleratedatherosclerosis. Thus, such subjects would benefit from theadministration of a polypeptide derived from sRAGE in an effectiveamount over an effective time.

The subject of the present invention may demonstrate clinical signs ofatherosclerosis, hypercholesterolemia or other disorders as discussedhereinbelow.

Clinically, hypercholesterolemia may be treated by interrupting theenterohepatic circulation of bile acids. It is reported that significantreductions of plasma cholesterol can be effected by this procedure,which can be accomplished by the use of cholestyramine resin orsurgically by the ileal exclusion operations. Both procedures cause ablock in the reabsorption of bile acids. Then, because of release fromfeedback regulation normally exerted by bile acids, the conversion ofcholesterol to bile acids is greatly enhanced in an effort to maintainthe pool of bile acids. LDL (low density lipoprotein) receptors in theliver are up-regulated, causing increased uptake of LDL with consequentlowering of plasma cholesterol.

Cholesterol, Atherosclerosis, and Coronary Heart Disease

Many investigators have demonstrated a correlation between raised serumlipid levels and the incidence of coronary heart disease andatherosclerosis in humans. Of the serum lipids, cholesterol has been theone most often singled out as being chiefly concerned in therelationship. However, other parameters—such as serum triacylglycerolconcentration—show similar correlations. Patients with arterial diseasecan have any one of the following abnormalities: (1) elevatedconcentrations of VLDL (very low density lipoproteins) with normalconcentrations of LDL; (2) elevated LDL with Normal VLDL; (3) elevationof both lipoprotein fractions. There is also an inverse relationshipbetween HDL (high density lipoproteins) (HDL₂) concentrations andcoronary heart disease, and some consider that the most predictiverelationship is the LDL:HDL cholesterol ratio. This relationship isexplainable in terms of the proposed roles of LDL in transportingcholesterol to the tissues and of HDL acting as the scavenger ofcholesterol.

Atherosclerosis is characterized by the deposition of cholesterol andcholesteryl eater of lipoproteins containing apo-B-100 in the connectivetissue of the arterial walls. Diseases in which prolonged elevatedlevels of VLDL, IDL, or LDL occur in the blood (e.g., diabetes,mellitus, lipid nephrosis, hypothyroidism, and other conditions ofhyperlipidemia) are often accompanied by premature or more severatherosclerosis.

Experiments on the induction of atherosclerosis in animals indicate awide species variation in susceptibility. The rabbit, pig, monkey, andhumans are species in which atherosclerosis can be induced by feedingcholesterol. The rat, dog, mouse and cat are resistant. Thyroidectomy ortreatment with thiouracil drugs will allow induction of atherosclerosisin the dog and rat. Low blood cholesterol is a characteristic ofhyperthyroidism.

Hereditary factors play the greatest role in determining individualblood cholesterol concentrations, but of the dietary and environmentalfactors that lower blood cholesterol, the substitution in the diet ofpolyunsaturated fatty acids for some of the saturated fatty acids hasbeen the most intensely studied.

Naturally occurring oils that contain a high proportion of linoleic acidare beneficial in lowering plasma cholesterol and include peanut,cottonseed, corn, and soybean oil whereas butterfat, beef fat, andcoconut oil, containing a high proportion of saturated fatty acids,raise the level. Sucrose and fructose have a greater effect in raisingblood lipids, particularly triacylglycerols, than do othercarbohydrates.

The reason for the cholesterol-lowering effect of polyunsaturated fattyacids is still not clear. However, several hypotheses have been advancedto explain the effect, including the stimulation of cholesterolexcretion into the intestine and the stimulation of the oxidation ofcholesterol to bile acids. It is possible that cholesteryl esters ofpolyunsaturated fatty acids are more rapidly metabolized by the liverand other tissues, which might enhance their rate of turnover andexcretion. There is other evidence that the effect if largely due to ashift in distribution of cholesterol from the plasma into the tissuesbecause of increased catabolic, rate of LDL. Saturated fatty acids causethe formation of smaller VLDL particles that contain relatively morecholesterol, and they are utilized by extrahepatic tissues at a slowerrate than are larger particles. All of these tendencies may be regardedas atherogenic.

Additional factors considered to play a part in coronary heart diseaseinclude high blood pressure, smoking, obesity, lack of exercise, anddrinking soft as opposed to hard water. Elevation of plasma free fattyacids will also lead to increase VLDL secretion by the liver, involvingextra triacylglycerol and cholesterol output into the circulation.Factors leading to higher or fluctuating levels of free fatty acidsinclude emotional stress, nicotine from cigarette smoking, coffeedrinking, and partaking of a few large meals rather than more continuousfeeding. Premenopausal women appear to be protected against many ofthese deleterious factors, possibly because they have higherconcentrations of HDL than do men and postmenopausal women.

Hypolipidemic Drugs

When dietary measures fail to achieve reduced serum lipid levels, theuse of hypolipidemic drugs may be resorted to. Several drugs are knownto block the formation of cholesterol at various stages in thebiosynthetic pathway. Many of these drugs have harmful effects, but thefungal inhibitors of HMG-CoA reductase, compactin and mevinolin, reduceLDL cholesterol levels with few adverse effects. Sitosterol is ahypocholesterolemic agent that acts by blocking the absorption ofcholesterol in the gastrointestinal tract. Resins such as colestipol andcholestyramine (Questran) prevent the reabsorption of bile salts bycombining with them, thereby increasing their fecal loss. Neomycin alsoinhibits reabsorption of bile salts. Clofibrate and gembivrozil exert atleast part of their hypolipidemic effect by diverting the hepatic flowof free fatty acids from the pathways of esterification into those ofoxidation, thus decreasing the secretion of triacylglycerol andcholesterol containing VLDL by the liver. In addition, they facilitatehydrolysis of VLDL triacylglycerols by lipoprotein lipase. Probucolappears to increase LDL catabolism via receptor-independent pathways.Nicotinic acid reduces the flux of FFA by inhibiting adipose tissuelipolysis, thereby inhibiting VLDL production by the liver.

Disorders of the Plasma Lipoproteins (Dyslipoproteinemias)

A few individuals in the population exhibit inherited defects in theirlipoproteins, leading to the primary condition of whether hypo- orhyperlipoproteinemia. Many others having defects such as diabetesmellitus, hypothyroidism, and atherosclerosis show abnormal lipoproteinpatterns that are very similar to one or another of the primaryinherited conditions. Virtually all of these primary conditions are dueto a defect at one or another stage in the course of lipoproteinformation, transport, or destruction. Not all of the abnormalities areharmful.

Hypolipoproteinemia:

1. Abetalipoproteinemia—This is a rare inherited disease characterizedby absence of β-lipoprotein (LDL) in plasma. The blood lipids arepresent in low concentrations—especially acylglycerols, which arevirtually absent, since no chylomicrons or VLDL are formed. Both theintestine and the liver accumulate acylglycerols. Abetalipoproteinemiais due to a defect in apoprotein B synthesis.

2. Familial hypobetalipoproteinemia—In hypobetalipoproteinemia, LDLconcentration is between 10 and 50% of normal, but chylomicron formationoccurs. It must be concluded that apo-B is essential for triacylglyceroltransport. Most individuals are healthy and long-lived.

3. Familial alpha-lipoprotein deficiency (Tangier disease)—In thehomozygous individual, there is near absence of plasma HDL andaccumulation of cholesteryl esters in the tissues. There is noimpairment of chylomicron formation or secretion of VLDL by the liver.However, on electrophoresis, there is no pre-β-lipoprotein, but a broadβ-band is found containing the endogenous triacylglycerol. This isbecause the normal pre-β-band contains other apo-proteins normallyprovided by HDL. Patients tend to develop hypertriacylglycerolemia as aresult of the absence of apo-C-II, which normally activates lipoproteinlipase.

Hyperlipoproteinemia:

1. Familial lipoprotein lipase deficiency (type I)—This condition ischaracterized by very slow clearing of chylomicrons from thecirculation, leading to abnormally raised levels of chylomicrons. VLDLmay be raised, but there is a decrease in LDL and HDL. Thus, thecondition is fat-induced. It may be corrected by reducing the quantityof fat and increasing the proportion of complex carbohydrate in thediet. A variation of this disease is caused by a deficiency in apo-C-II,required as a cofactor for lipoprotein lipase.

2. Familial hypercholesterolemia (type II)—Patients are characterized byhyperbetalipoproteinemia (LDL), which is associated with increasedplasma total cholesterol. There may also be a tendency for the VLDL tobe elevated in type IIb. Therefore, the patient may have somewhatelevated triacylglycerol levels but the plasma—as is not true in theother types of hyperlipoproteinemia—remains clear. Lipid deposition inthe tissue (e.g., xanthomas, atheromas) is common. A type II pattern mayalso arise as a secondary result of hypothyroidism. The disease appearsto be associated with reduced rates of clearance of LDL from thecirculation due to defective LDL receptors and is associated with anincreased incidence of atherosclerosis. Reduction of dietary cholesteroland saturated fats may be of use in treatment. A disease producinghypercholesterolemia but due to a different cause is Wolman's disease(cholesteryl ester storage disease). This is due to a deficiency ofcholesteryl ester hydrolase in lysosomes of cells such as fibroblaststhat normally metabolize LDL.

3. Familial type III hyperlipoproteinemia (broad beta disease, remnantremoval disease, familial dysbetalipoproteinemia)—This condition ischaracterized by an increase in both chylomicron and VLDL remnant; theseare lipoproteins of density less than 1.019 but appear as a broad β-bandon electrophoresis (β-VLDL). They cause hypercholesterolemia andhypertriacylglycerolemia. Xanthomas and atherosclerosis of bothperipheral and coronary arteries are present. Treatment by weightreduction and diets containing complex carbohydrates, unsaturated fats,and little cholesterol is recommended. The disease is due to adeficiency in remnant metabolism by the liver caused by an abnormalityin apo-E, which is normally present in 3 isoforms, E2, E3, and E4.Patients with type III hyperlipoproteinemia possess only E2, which doesnot react with the E receptor.

4. Familial hypertriacylglycerolemia (type IV)—This condition ischaracterized by high levels of endogenously producedtriacylglycerol(VLDL). Cholesterol levels rise in proportion to thehypertriacylglycerolemia, and glucose intolerance is frequently present.Both LDL and HDL are subnormal in quantity. This lipoprotein pattern isalso commonly associated with coronary heart disease, type IInon-insulin-dependent diabetes mellitus, obesity, and many otherconditions, including alcoholism and the taking of progestationalhormones. Treatment of primary type IV hyperlipoproteinemia is by weightreduction; replacement of soluble diet carbohydrate with complexcarbohydrate, unsaturated fat, low-cholesterol diets; and alsohypolipidemic agents.

5. Familial type V hyperlipoproteinemia—The lipoprotein pattern iscomplex, since both chylomicrons and VLDL are elevated, causing bothtriacylglycerolemia and cholesterolemia. Concentrations of LDL and HDLare low. Xanthomas are frequently present, but the incidence ofatherosclerosis is apparently not striking. Glucose tolerance isabnormal and frequently associated with obesity and diabetes. The reasonfor the condition, which is familial, is not clear. Treatment hasconsisted of weight reduction followed by a diet not too high in eithercarbohydrate or fat.

It has been suggested that a further cause of hypolipoproteinemia isoverproduction of apo-B, which can influence plasma concentrations ofVLDL and LDL.

6. Familial hyperalphalipoproteinemia—This is a rare conditionassociated with increased concentrations of HDL apparently beneficial tohealth.

Familial Lecithin: Cholesterol Acyltransferase (LCAT) Deficiency: Inaffected subjects, the plasma concentration of cholesteryl esters andlysolecithin is low, whereas the concentration of cholesterol andlecithin is raised. The plasma tends to be turbid. Abnormalities arealso found in the lipoproteins. One HDL fraction contains disk-shapedstructures in stacks or rouleaux that are clearly nascent HDL unable totake up cholesterol owing to the absence of LCAT. Also present as anabnormal LDL subfraction is lipoprotein-X, otherwise found only inpatients with cholestasis. VLDL are also abnormal, migrating asβ-lipoproteins upon electrophoresis (β-VLDL). Patients with parenchymalliver disease also show a decrease of LCAT activity and abnormalities inthe serum lipids and lipoproteins.

Pharmaceutical with Carriers

In one preferred embodiment the pharmaceutical carrier may be a liquidand the pharmaceutical composition would be in the form of a solution.In another equally preferred embodiment, the pharmaceutically acceptablecarrier is a solid and the composition is in the form of a powder ortablet. In a further embodiment, the pharmaceutical carrier is a gel andthe composition is in the form of a suppository or cream. In a furtherembodiment the active ingredient may be formulated as a part of apharmaceutically acceptable transdermal patch.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by for example, intramuscular, intrathecal,epidural, intraperitoneal or subcutaneous injection. Sterile solutionscan also be administered intravenously. The active ingredient may beprepared as a sterile solid composition which may be dissolved orsuspended at the time of administration using sterile water, saline, orother appropriate sterile injectable medium. Carriers are intended toinclude necessary and inert binders, suspending agents, lubricants,flavorants, sweeteners, preservatives, dyes, and coatings.

The active ingredient of the present invention (i.e., polypeptidederived from sRAGE, or composition) can be administered orally in theform of a sterile solution or suspension containing other solutes orsuspending agents, for example, enough saline or glucose to make thesolution isotonic, bile salts, acacia, gelatin, sorbitan monoleate,polysorbate 80 (oleate esters of sorbitol and its anhydridescopolymerized with ethylene oxide) and the like.

The active ingredient can also be administered orally either in liquidor solid composition form. Compositions suitable for oral administrationinclude solid forms, such as pills, capsules, granules, tablets, andpowders, and liquid forms, such as solutions, syrups, elixirs, andsuspensions. Forms useful for parenteral administration include sterilesolutions, emulsions, and suspensions.

Atherosclerosis

In one embodiment of the present invention, the subject may bepredisposed to atherosclerosis. This predisposition may include geneticpredisposition, environmental predisposition, metabolic predispositionor physical predisposition. There have been recent reviews ofatherosclerosis and cardiovascular disease. For example: Keating andSanguinetti, (May 1996) Molecular Genetic Insights into CardiovascularDisease, Science 272:681-685 is incorporated by reference in itsentirety into the present application. The authors review theapplication of molecular tools to inherited forms of cardiovasculardisease such as arrhythmias, cardiomyopathies, and vascular disease.Table 1 of this reference includes cardiac diseases and the aberrantprotein associated with each disease. The diseases listed are: LQTdisease, familial hypertrophic cardiomyopathy; duchenne and Beckermuscular dystrophy; Barth syndrome Acyl-CoA dehydrogenase deficiencies;mitochondrial disorders; familial hypercholesterolemia;hypobetalipoproteinemia; homocystinuria; Type III hyperlipoproteinemia;supravalvular aortic stenosis; Ehler-Danlos syndrome IV; Marfa syndrome;Heredity hemorrhagic telangiectasia. These conditions are included aspossible predispositions of a subject for atherosclerosis.

Furthermore, mouse models of atherosclerosis are reviewed in Breslow(1996) Mouse Models of Atherosclerosis, Science 272:685. This referenceis also incorporated by reference in its entirety into the presentapplication. Breslow also includes a table (Table 1) which recitesvarious mouse models and the atherogenic stimulus. For example, mousemodels include C57BL/6; Apo E deficiency; ApoE lesion; ApoE R142C; LDLreceptor deficiency; and HuBTg. One embodiment of the present inventionis wherein a subject has a predisposition to atherosclerosis as shown bythe mouse models presented in Breslow's publication.

Gibbons and Dzau review vascular disease in Molecular Therapies forVascular Disease, Science Vol. 272, pages 689-693. In one embodiment ofthe present invention, the subject may manifest the pathological eventsas described in Table 1 of the Gibbons and Dzau publication. Forexample, the subject may have endothelial dysfunction, endothelialinjury, cell activation and phenotypic modulation, dysregulated cellgrowth, dysregulated apoptosis, thrombosis, plaque rupture, abnormalcell migration or extracellular or intracellular matrix modification.

In another embodiment of the present invention, the subject may havediabetes. The subject may demonstrate complications associated withdiabetes. Some examples of such complications include activation ofendothelial and macrophage AGE receptors, altered lipoproteins, matrix,and basement membrane proteins; altered contractility and hormoneresponsiveness of vascular smooth muscle; altered endothelial cellpermeability; sorbitol accumulation; neural myoinositol depletion oraltered Na-K ATPase activity. Such complications are discussed in arecent publication by Porte and Schwartz, Diabetes Complications: Why isGlucose potentially Toxic?, Science, Vol. 272, pages 699-700.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

Experimental Details

EXAMPLE 1 Suppression of Accelerated Diabetic Athroaclaerosis by SolubleReceptor for Advanced Glycation Endproducts (sRAGE)

Central to diabetes is the presence of hyperglycemia. An importantcomplication of the interaction of glucose with proteins/lipids isirreversible formation of Advanced Glycation Endproducts, or AGEs(Brownlee, 1992). AGEs accumulate in the plasma and tissues duringnormal aging, and to an accelerated degree in patients with diabetes.AGEs have been linked to the pathogenesis of diabetic complications.

It has been demonstrated that interaction of AGEs with their cellularreceptor RAGE Receptor for AGE) on monocytes and endothelial cellsresults in the development of a proinflammatory environment in whichenhanced monocyte migration/activation, endothelial hyperpermeability,and enhanced expression of adhesion molecules and tissue factor onendothelial cells results in the generation of an environment conduciveto the development of vascular lesions (Schmidt et al, 1992; Neeper etal, 1992; Schmidt et al, 1994; Schmidt et al, 1995).

It has also been demonstrated that the extracellular portion of RAGE(called soluble or sRAGE), composed of one “V”-type immunoglobulindomain followed by two “C”-type domains interferes with the ability ofAGEs to bind to and activate cell RAGE (Schmidt et al., 1994). In vivo,administration of sRAGE blocks hyperpermeabilty in diabetic rats(Wautier et al., 1996).

As discussed hereinbelow, a model of accelerated atherosclerosis indiabetes was developed and used to test the hypothesis that chronicadministration of sRAGE (to/2 elimination in diabetic rodents of 22 hrs)prevents the development of accelerated atherosclerosis.

These studies demonstrated that daily intraperitoneal injection of sRAGEprevents the accelerated development of atherosclerosis inapolipoprotein E deficient (or knockout) mice rendered diabetic withstreptozotocin.

Materials and Methods:

Animals and the induction of diabetes. Apolipoprotein E(0) mice on theC57B1/6J background (N10; 10 generations backcrossed with >99% homology)were obtained from the Jackson Laboratories. At the age of 7 wks,diabetes was induced in certain male mice with multiple intraperitonealinjections of streptozotocin (55 mg/kg) in 4 daily injections in sterilecitrate buffer (0.05M; pH 4.5). Control mice were treated with vehicle(buffer alone). Plasma glucose concentration was then determined withcolormetric assay (Sigma®) using blood obtained from the tail vein. Micewere considered diabetic if plasma glucose levels exceeded 300 mg/dl ontwo separate occasions. All mice were maintained on normal chow diet.

Quantitation of atherosclerotic lesions. Mice were sacrificed at thetime points indicated below after induction of diabetes or controltreatment. Quantitative analysis of atherosclerotic lesions wasperformed on sections from the aortic sinus. After humane sacrifice,hearts were fixed in formalin (10%), embedded in gelatin (25%) andfrozen. Cryostat sections were cut 10 microns thick, stained with oilred O and counterstained with hematoxylin and light green. Fatty lesionarea was then determined by computer assisted image analysis (ZeissImage, Media Cybernetics) in five consecutive sections, each separatedby 80 microns. Mean lesion area was quantitated for each group.

Preparation of soluble mouse RAGE. A construct containing mouse solubleRAGE cDNA was prepared and co-transfected with baculovirus DNA accordingto the manufacturer's instructions (PharMingen). Sf9 cells were theninfected with the construct for three days in Grace's insect mediumcontaining fetal bovine serum (10%), followed by three days in Grace'sinsect medium without serum (Gibco®). At the end of the second threedays, cells were separated from supernatant using centrifugation (1,200rpm×20 mins) and supernatant dialyzed versus buffer containing sodiumphosphate (0.02M; pH 7.4) and NaCl (0.05M). After dialysis, supernatantwas applied to an SP sepharose resin (5 ml) using the FPLC system(Pharmacia®). Mouse soluble RAGE was eluted using a linear gradient ofsodium chloride (0.05M to 0.6M). SDS-PAGE revealed the material to besingle-band. Prior to introduction into mice, purified mouse solubleRAGE was passed through an endotoxin-removal column (Detoxigel, Pierce).Final product was devoid of endotoxin as determined by testing in thelimulus amebocyte assay (Sigma®), dialyzed versus phosphate-bufferedsaline and stored in aliquots at 80° C.

Treatment of diabetic mice with soluble RAGE. After induction ofdiabetes, certain diabetic mice were treated once daily with eithersoluble mouse RAGE (20 μg/day; intraperitoneally) or with equimolarconcentrations of mouse serum albumin (40 μg/day; intraperitoneally)beginning two weeks after induction of diabetes and continuing for sixwks. At the end of that time, mice were sacrificed and the aortasubjected to quantitative morphometric analysis of atheroscleroticlesions.

Analysis of lipoproteins. Mice were fasted four hours prior to obtainingplasma for analysis of lipoproteins. Plasma concentrations ofcholesterol and triglyceride were measured using commercial kits(Boehringer Mannheim®). VLDL (very low density lipoproteine), IDL(intermediate density lipoproteins)/LDL (low density lipoproteins), andHDL (high density lipoproteins) were separated by densityultracentrifugation as well as by FPLC.

Results

After treatment with streptozotocin (stz), mean plasma glucoseconcentration was approximately 350-500 mg/dl, compared with 130-160mg/dl in controls.

Consistent with previous studies in apo E(0) mice (Plump et al., 1992),fatty streak lesions were observed in both diabetic and control mice atthe early time points (4 wks). The lesions first appeared at the aorticroot and at the lesser curvature of the arch of the aorta, withprogression to each of the principal branches of the thoracic aorta,beginning proximally. At each time point, the lesions were consistentlylarger in size and more extensive in the diabetic mice compared with thecontrols. For example, after ten weeks of diabetes, mice demonstrateddiscrete lesions at each of the thoracic branch points, with nearlycomplete occlusion of the vessels (FIG. 1B). This was in marked contrastto the age-matched, citrate-treated control mice, in whom only mildfatty streaks, mostly at the aortic root were visualized (FIG. 1A).

Quantitative analysis of the lesions revealed that after eight weeks ofdiabetes, mean lesion area in the diabetic mice wan approximately3.7-fold higher than that observed in the nondiabetic controls.Visualization with oil red O/hematoxylin light green demonstratedadvanced atherosclerotic lesions with evidence of fibrous cap formationafter 8 weeks of diabetes. A similar experiment revealed anapproximately 3-fold increase in mean lesion area after 6 wks ofdiabetes.

Analysis of lipid profile indicated that induction of diabetes resultedin an approximately 2-fold increase in the levels of VLDL, anapproximately 1.4-fold increase in the Levels of LDL and no change inthe levels of HDL, compared with citrate-treated control mice. Therewere no differences in levels of plasma triglyceride between diabeticand nondiabetic mice.

Consistent with the hypothesis as presented herein that enhancedAGE-RAGE interaction was important in the pathogenesis of acceleratedatherosclerosis in diabetic mice, treatment of diabetic mice with sRAGE(20 μ/day; intraperitoneally) resulted in an approximately 1.8-folddecrease in mean lesion area compared with diabetic mice treated withmouse serum albumin, 150,046±18,549 vs. 271,008±16,721 μm²,respectively, p<0.02 (FIG. 2). Visual inspection of the aortic tree of atypical diabetic mouse after 8 weeks of diabetes treated with mouseserum albumin revealed evidence of extensive atherosclerotic plaques atthe major branch points and at the arch of the aorta (FIG. 3A), whichwere markedly diminished in diabetic mice treated with sRAGE (FIG. 3B).Importantly, mice treated with sRAGE demonstrated no alteration in theirlevels of plasma glucose. Furthermore, mice treated with sRAGEmanifested no differences in lipid profile (total cholesterol, totaltriglyceride, as well as fractionation of lipoproteins by FPLCanalysis), compared with diabetic mice treated with mouse serum albumin.These data suggest that treatment with sRAGE diminished accelerateddiabetic atherosclerosis in a glucose- and lipid-independent manner.

Discussion

As detailed herein, the development of one of the first models ofaccelerated atherosclerosis in a diabetic mouse after treatment withstreptozotocin has been demonstrated. Earlier and more advancedatherosclerotic lesions were demonstrated in diabetic mice as comparedwith age-matched controls.

An important role for enhanced AGE-RAGE interaction in the developmentof accelerated diabetic atherosclerosis, treatment of diabetic mice withsRAGE, a competitive inhibitor of the interaction of AGEs with cellularRAGE, resulted in a statistically-significant decrease in meanatherosclerotic lesion area after 8 weeks of diabetes.

Taken together, these data indicate that administration of soluble RAGEmay be a new and important means by which to prevent chroniccomplications of diabetes, such as accelerated atherosclerosis.

REFERENCES

-   Brownlee M. Glycation products and the pathogenesis of diabetic    complications. Diab. Care 15(12) :1835-1842, 1992.-   Carpenter, et al. (1971) Toxicol. Appl. Pharmacol., 18:35-40.-   Crall F V J and W C Roberts. The extramural and intramural coronary    arteries in juvenile diabetes mellitus: analysis of nine necropsy    patients aged 19 to 38 years with onset of diabetes before age 15    years. Am. J. Med. 64:221-230, 1978.-   Hamby R I et al. Reappraisal of the role of the diabetic state in    coronary artery disease. Chest 2:251-257, 1976.-   Kannel W B and D L McGee. Diabetes and cardiovascular disease: the    Framingham study. J. Am. Med. Assoc.241:2035-2038, 1979.-   Kannel W B and D L McGee. Diabetes and glucose tolerance as risk    factors for cardiovascular disease: the Framingham study. Diab. Care    2:120-126, 1979.-   Manson J E et al. A prospective study of maturity-onset diabetes    mellitus and risk of coronary heart disease and stroke in women.    Arch. Of. Int. Med. 151:1141-1137, 1991.-   Neeper M, A M Schmidt, J Brett, S D Yan, F Wang, Y C Pan, K    Elliston, D Stern and A Shaw. Cloning and expression of RAGE: a cell    surface receptor for advanced glycocylation end products of    proteins. J. Biol. Chem. 267:14998-15004, 1992.-   Plump, A S et al. Severe hypercholesterolemia and atherosclerosis in    apolipoprotein E-deficient mice created by homologous recombination    in ES cells. Cell 71:343-353, 1992.-   Pyorala K M, M Laasko and M Uusitupa. Diabetes and atherosclerosis:    an epidemiologic view. Diab. Metab. Rev. 3:463-524, 1987.-   Robertson W B and J B Strong. Atherosclerosis in persons with    hypertension and diabetes mellitus. Lab. Invest. 18: 538-551, 1968.-   Schmidt A M, M Vianna, M Gerlach, J Brett, J Ryan, J Kao, C    Esposito, H Hegarty, W Hurley, M Clause, P Wang, Y C Pan, T C Tsang,    and D Stern. Isolation and characterization of binding proteins for    advanced glycosylation end products from lung tissue which are    present on the endothelial cell surface. J. Biol. Chem.    267:14987-14997, 1992.-   Schmidt A M, O Hori, J Brett, S D Yan, J L Wautier, and D Stern.    Cellular receptors for advanced glycation endproducts: implications    for induction of oxidant stress and cellular dysfunction in the    pathogenesis of vascular lesions. Arterioscl. And Thromb.    14:1521-1528, 1994.-   Schmidt A M, S D Yan, and D Stern. The dark side of glucose(News and    Views). Nat. Med. 1:1002-1004, 1995.-   Schmidt A M, M Hasu, D Popov, J H Zhang, S D Yan, J Brett, R Cao, K    Kuwabara, G Costache, N Simionescu, and D Stern. The receptor for    Advanced Glycation Endproducts(RAGE) has a central role in vessel    wall interactions and gene activation in response to AGEs in the    intravascular space. PNAS(USA) 91:8807-8811, 1994.-   Waller B F et al. Status of the coronary arteries at necropsy in    diabetes mellitus with onset after age 30 yrs: analysis of 229    diabetic patients with and without clinical evidence of coronary    heart disease and comparison to 183 control subjects. Am. J. Med.    69:498-506, 1980.-   Wautier J L, C Zoukourian, O Chappey, M P Wautier, P J Guillausseau,    R Cao, O Hori, D Stern, and A M Schmidt. Recepter-mediated    endothelial cell dysfunction in diabetic vasculopathy: soluble    receptor for advanced glycation endproducts blocks    hyperpermeability. J. Clin. Invest. 97:238-243, 1996.

1-35. (canceled)
 36. A method for preventing accelerated atherosclerosisin a subject afflicted with hypercholesterolemia which comprisesadministering to the subject a polypeptide comprising a solubleextracellular portion of a receptor for advanced glycation endproduct(sRAGE) capable of inhibiting an interaction between amyloid-β peptideand receptor for advanced glycation endproduct (RAGE) in an amounteffective to prevent accelerated atherosclerosis in the subject.
 37. Themethod of claim 36, wherein the subject is a mammal.
 38. The method ofclaim 37, wherein the mammal is a human.
 39. The method of claim 36,wherein the polypeptide comprises a V domain of naturally occurringsoluble receptor for advanced glycation endproduct.
 40. The method ofclaim 36, wherein the polypeptide comprises a 10 kilodalton domain ofnaturally occurring soluble receptor for advanced glycation endproduct.41. The method of claim 36, wherein the polypeptide comprises a sequenceless than or equal to 20 amino acids in length which sequence is withinthe sequence for naturally occurring soluble receptor for advancedglycation endproduct.
 42. The method of claim 36, further comprisingadministering to the subject a pharmaceutically acceptable carrierduring the administration of the polypeptide.
 43. The method of claim36, wherein the administration comprises intralesional, intraperitoneal,intramuscular or intravenous injection; infusion; liposome-mediateddelivery; or topical, nasal, oral, ocular or otic delivery.
 44. Themethod of claim 36, wherein the polypeptide is administered daily. 45.The method of claim 36, wherein the effective amount of the polypeptidecomprises from about 0.000001 mg/kg body weight to about 100 mg/kg bodyweight.
 46. A method for preventing accelerated atherosclerosis in asubject afflicted with hypertriglyceridemia which comprisesadministering to the subject a polypeptide comprising a solubleextracellular portion of a receptor for advanced glycation endproduct(sRAGE) capable of inhibiting an interaction between amyloid-β peptideand receptor for advanced glycation endproduct (RAGE) in an amounteffective to prevent accelerated atherosclerosis in the subject.
 47. Themethod of claim 46, wherein the subject is a mammal.
 48. The method ofclaim 47, wherein the mammal is a human.
 49. The method of claim 46,wherein the polypeptide comprises a V domain of naturally occurringsoluble receptor for advanced glycation endproduct.
 50. The method ofclaim 46, wherein the polypeptide comprises a 10 kilodalton domain ofnaturally occurring soluble receptor for advanced glycation endproduct.51. The method of claim 46, wherein the polypeptide comprises a sequenceless than or equal to 20 amino acids in length which sequence is withinthe sequence for naturally occurring soluble receptor for advancedglycation endproduct.
 52. The method of claim 46, further comprisingadministering to the subject a pharmaceutically acceptable carrierduring the administration of the polypeptide.
 53. The method of claim46, wherein the administration comprises intralesional, intraperitoneal,intramuscular or intravenous injection; infusion; liposome-mediateddelivery; or topical, nasal, oral, ocular or otic delivery.
 54. Themethod of claim 46, wherein the polypeptide is administered daily. 55.The method of claim 46, wherein the effective amount of the polypeptidecomprises from about 0.000001 mg/kg body weight to about 100 mg/kg bodyweight.